All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.
Glutamate is an amino acid and one type of neurotransmitter found in the brain. Glutamatergic neurons are the predominant excitatory pathways in the mammalian brain, representing one-third of all rapid excitatory synapses in the central nerve system (Cotman, C. W., and Monaghan, D. T. (1986) Anatomical organization of excitatory amino acid receptors and their properties. Adv. Exp. Med. Biol. 203, 237-252). Signaling by glutamate is mediated by a large and diverse number of receptors, including ionotropic receptors that allow passage of extracellular calcium through coupled ion channels upon activation, and metabotropic receptors that activate intermediary molecules such as G proteins to produce molecules such as IP3 that increase cytosolic calcium concentrations. Interaction between neurons may be either excitatory or inhibitory. The major excitatory amino acid neurotransmitters are glutamate and aspartate, while GABA (γ-aminobutyric acid), glycine (aminoacetic acid), and taurine are inhibitory (Mark et al. (2001) American Journal of Neuroradiology 22:1813-1824).
Clearance of extracellular glutamate by glutamate transporters is an indispensable step to prevent the accumulation of glutamate, which would otherwise result in overstimulation of glutamate receptors and glutamate excitotoxicity. Excitotoxic damage causes, or is involved in, a number of neurologic diseases, including stroke, trauma, epilepsy, and neurodegenerative conditions, such as Huntington disease, AIDS dementia complex, and amyotrophic lateral sclerosis (Doble, A., Louvel, E., and Hugon, J. (1999) The role of excitotoxicity in neurodegenerative disease: implications for therapy, Pharmacol. Ther. 81(3): 163-221; Waggie K S, Kahle P J, Tolwani R J. (1999) Neurons and mechanisms of neuronal death in neurodegenerative diseases: a brief review. Lab. Anim. Sci. 49:358-362). Glutamate receptor overstimulation increases intracellular calcium by directly opening ion channels, allowing the influx of calcium and causing membrane depolarization. Depolarization in turn activates voltage-dependent calcium channels, which further increases the intracellular calcium levels. The glutamate-induced elevated calcium levels causes overactivation of a number of enzymes, including protein kinase C, calcium/calmodulin-dependent protein kinase II, phospholipases, proteases, phosphatases, nitric oxide synthase, endonucleases, and ornithine decarboxylase, some of which produce toxic free oxygen radicals, or produce positive feedback loops leading to neuronal death (Mark et al., 2001).
The key factor that triggers the excitotoxic cascade is the excessive accumulation of glutamate in the synaptic space. Normal extracellular glutamate concentration is about 0.6 μmol/L, with substantial neuronal excitotoxic injury occurring at glutamate concentrations of 2 to 5 μmol/L. Traumatic injury to neurons can produce disastrous results with the release of about 10 μmol/L to the extracellular space. Given the ensuing cascade, injury to a single neuron puts all of the neighboring neurons at risk (Mark et al., 2001).
Despite a number of studies showing the involvement of higher glutamate concentration in neurologic diseases, measuring glutamate concentration in living cells remains challenging. One of the most important tools required to assign functions of neurons in vivo would be to visualize glutamate fluxes directly. The extracellular concentration of glutamate has been measured by in vivo microdialysis techniques (Faden, A. I., Demediuk, P., Panter, S. S., and Vink, R. (1989) The role of excitatory amino acids and NMDA receptors in traumatic brain injury. Science 244, 798-800; Fallgren, A. B., and Paulsen, R. E. (1996) A microdialysis study in rat brain of dihydrokainate, a glutamate uptake inhibitor. Neurochem Res 21, 19-25). However, microdialysis is limited in spatial and temporal resolution, unable to detect the localized and rapid concentration change around a single synapse. In addition, the in vivo microdialysis technique is destructive. It also does not permit direct monitoring of glutamate levels inside living neurons or astrocytes.
In vivo measurement of ions and metabolites by using Fluorescence Resonance Energy Transfer (FRET) has been successfully used to measure calcium concentration changes, by fusing CFP, YFP, and a reporter domain consisting of calmodulin and the M13 peptide (Zhang, J., Campbell, R. E., Ting, A. Y., and Tsien, R. Y. (2002a) Creating new fluorescent probes for cell biology. Nat Rev Mol Cell Biol 3, 906-918; Zhang, J., Campbell, R. E., Ting, A. Y., and Tsien, R. Y. (2002b) Creating new fluorescent probes for cell biology. Nature Reviews Molecular Cell Biology 3, 906-918). Binding of calcium to calmodulin causes global structural rearrangement of the chimera resulting in a change in FRET intensity as mediated by the donor and acceptor fluorescent moieties. Recently a number of bacterial periplasmic binding proteins, which undergo a venus flytrap-like closure of two lobes upon substrate binding, have been successfully used as the scaffold of metabolite nanosensors (Fehr, M., Frommer, W. B., and Lalonde, S. (2002) Visualization of maltose uptake in living yeast cells by fluorescent nanosensors. Proc. Natl. Acad. Sci. USA 99, 9846-9851; Fehr, M., Lalonde, S., Lager, I., Wolff, M. W., and Frommer, W. B. (2003) In vivo imaging of the dynamics of glucose uptake in the cytosol of COS-7 cells by fluorescent nanosensors. J. Biol. Chem. 278, 19127-19133; Lager, I., Fehr, M., Frommer, W. B., and Lalonde, S. (2003) Development of a fluorescent nanosensor for ribose. FEBS Lett 553, 85-89).
In order to develop a nanosensor protein for glutamate, we searched for a protein which changes conformation upon binding glutamate. The family of ionotropic (iGluRs) and metabotropic glutamate receptor (mGluRs) have an extracellular ligand binding domain which has sequence similarity to bacterial periplasmic binding proteins (O'Hara, P. J., Sheppard, P. O., Thogersen, H., Venezia, D., Haldeman, B. A., McGrane, V., Houamed, K. M., Thomsen, C., Gilbert, T. L., and Mulvihill, E. R. (1993) The ligand-binding domain in metabotropic glutamate receptors is related to bacterial periplasmic binding proteins. Neuron 11, 41-52), as well as the ligand binding domain of γ-aminobytyric acid (GABA)B receptor (Kaupmann, K., Huggel, K., Heid, J., Flor, P. J., Bischoff, S., Mickel, S. J., McMaster, G., Angst, C., Bittiger, H., Froestl, W., and Bettler, B. (1997) Expression cloning of GABA(B) receptors uncovers similarity to metabotropic glutamate receptors. Nature 386, 239-246). The crystal structures of mGluR1 ligand binding domain in three different forms, in a complex with glutamate and in two unliganded forms, has been determined (Kunishima, N., Shimada, Y., Tsuji, Y., Sato, T., Yamamoto, M., Kumasaka, T., Nakanishi, S., Jingami, H., and Morikawa, K. (2000). Structural basis of glutamate recognition by a dimeric metabotropic glutamate receptor. Nature 407, 971-977), and the results suggested that glutamate binding stabilizes the closed conformation. Galvez et al. suggested that the ligand binding domain of GABAB receptor also undergoes the closure of two lobes (Galvez, T., Parmentier, M. L., Joly, C., Malitschek, B., Kaupmann, K., Kuhn, R., Bittiger, H., Froestl, W., Bettler, B., and Pin, J. P. (1999). Mutagenesis and modeling of the GABAB receptor extracellular domain support a venus flytrap mechanism for ligand binding. J Biol Chem 274, 13362-13369). We therefore attempted to construct FRET biosensors using the mGluR and GABAB receptors and assayed for changes in FRET efficiency upon addition of substrates. However, no change in FRET efficiency was observed. Similarly, also the LIV leucine/isoleucine/valine amino acid binding protein from bacteria could not be engineered into a functional FRET sensor.
De Lorimier et al. have shown that the YbeJ protein from E. coli, which shares sequence homology to glutamine- and histidine-binding proteins, and which is located in an operon involved in glutamate metabolism, binds to glutamate and aspartate (de Lorimier, R. M., Smith, J. J., Dwyer, M. A., Looger, L. L., Sali, K. M., Paavola, C. D., Rizk, S. S., Sadigov, S., Conrad, D. W., Loew, L., and Hellinga, H. W. (2002) Construction of a fluorescent biosensor family. Protein Sci 11, 2655-2675). The similarity of YbeJ to glutamine and histidine binding proteins from bacteria lead us to generate homology models based on the solved crystal structures of these two proteins. The 3D structure of the glutamine and histidine binding proteins indicates that N- and C-termini of these proteins are located on the same lobe, therefore the closure of two lobes upon substrate binding is unlikely to change the distance between N- and C-terminus. Thus none of these proteins should permit the construction of a FRET sensor on the same principle. Indeed, the sensors proposed by Hellinga and Looger in published U.S. patent application 20040118681 propose conjugating a single fluorophore to a cysteine residue that responds to a conformational change upon ligand binding, in contrast to the dual fluorescent moieties used for FRET.
Nevertheless, the present inventors have surprisingly found that the YbeJ protein of E. coli is an efficient FRET scaffold for detecting glutamate binding, despite the finding that both termini are located on the same lobe of the protein. This is in contrast to the general hypothesis that distance changes are converted to FRET changes.